Antioxidants 2014, 3, 516-525; doi:10.3390/antiox3030516 OPEN ACCESS antioxidants ISSN 2076-3921 www.mdpi.com/journal/antioxidants Article Phytochemicals and Antioxidative Properties of Borneo Indigenous Liposu (Baccaurea lanceolata) and Tampoi (Baccaurea macrocarpa) Fruits Mohd Fadzelly Abu Bakar 1,2,*, Nor Ezani Ahmad 1, Fifilyana Abdul Karim 1 and Syazlina Saib 1 1 Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu, Sabah 88400, Malaysia; E-Mails: [email protected] (N.E.A.); [email protected] (F.A.K.); [email protected] (S.S.) 2 Faculty of Science, Technology and Human Development, Universiti Tun Hussein Onn Malaysia (UTHM), Parit Raja, Batu Pahat, Johor 86400, Malaysia * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +6-088-320104; Fax: +6-088-320291. Received: 11 April 2014; in revised form: 16 June 2014 / Accepted: 23 June 2014 / Published: 30 July 2014 Abstract: Two underutilized indigenous fruits of Borneo, Liposu (Baccaurea lanceolata) and Tampoi (Baccaurea macrocarpa) were investigated for their total phenolic (TPC), flavonoid (TFC), anthocyanin (TAC) and carotenoid (TCC) contents as well as antioxidant properties in vitro. The fruits were separated into three different parts (i.e., pericarp, flesh and seed) and extracted using 80% methanol. Antioxidant activity was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging, ABTS decolorization and FRAP (Ferric Reducing Antioxidant Power) assays. The results showed that B. macrocarpa pericarp contained the highest amount of total phenolics, total flavonoid, total anthocyanin and total carotenoid with the values of 60.04 ± 0.53 mg GAE/g, 44.68 ± 0.67 mg CE/g, 1.23 ± 0.20 mg c-3-gE/100 g and 0.81 ± 0.14 mg BCE/g. Results from DPPH, ABTS and FRAP assays also showed that the pericarp of B. macrocarpa displayed the highest antioxidant capacity. The antioxidant activity of the extract was significantly correlated with the total phenolic and flavonoid contents, but not with the carotenoid contents. In conclusion, B. macrocarpa displayed high potential as natural source of phytochemicals with antioxidant properties. Antioxidants 2014, 3 517 Keywords: Baccaurea lanceolata; Baccaurea macrocarpa; phenolic; flavonoid; anthocyanin; carotenoid; antioxidant activity 1. Introduction Exogenous antioxidants are essential in the human body due to depletion of natural antioxidants [1]. They prevent or delay oxidation when exposed to free radicals and reactive oxygen species which are generated continuously inside the human body [2]. Reactive oxygen species might cause oxidative damage that leads to chronic diseases such as cancer, neurodegenerative disease, arthritis and diabetes mellitus [3]. Niki [4] reported that antioxidants can be evaluated by many methods but often give inconclusive and conflicting results. Therefore, assessment of antioxidants using more than one method is suggested to give more consistent results [5]. Previous studies have reported that high consumption of fresh fruits and vegetables may have protective effects against a wide range of chronic diseases caused by oxidative stress in the body [6]. This is mainly due to the presence of antioxidants constituents and bioactive compounds such as vitamin C, carotenoid, phenolics, flavonoids, tannins and anthocyanidins that are able to scavenge free radicals and inhibit lipid peroxidation [7–9]. A comprehensive analysis conducted based on the epidemiological studies available in the literature shows that there is undoubted evidence that higher consumption of fruits and vegetables reduces the risk of cardiovascular disease as well as cancer [10–12]. Despite many research on commonly consumed fruits, little information is available for underutilized indigenous fruits. Underutilized fruit is defined as fruit that is less popular, with under-exploited potential and not ready for commercialization [13]. Borneo (Malaysia—Sabah and Sarawak; Indonesia—Kalimantan and Brunei) has more than hundred types of indigenous fruits that can be found in backyards, small-scale orchards and also tropical rainforests. However, many of the indigenous fruits in Borneo are considered underutilized since they are only available locally and not much research has been conducted on these fruits. These underutilized fruits may contain a significant amount of phytochemicals or other unique compound which might have health-promoting properties. Their antioxidant capacity may be comparable or even superior to that of the more extensively studied fruits [14]. Baccaurea macrocarpa and Baccaurea lanceolata come from the genera of Baccaurea (family: Euphorbiaceae). Most of the Baccaurea species are endemic to Borneo. Both B. macrocarpa and B. lanceolata fruits are green to purple when young, and turn into yellow to orange when ripe. Both species are also dioecious, having the male and female reproductive organs borne on separate individuals of the same species, and can be propagate by seed. Although both species almost resembles each other, unlike B. lanceolata, B. macrocarpa trees have buttresses. B. lanceolata usually have one to four seeded berries with globose to ellipsoid fruit shape while the B. macrocarpa only have two to three seed berries with oblongoid to globose fruit shape [15]. For B. lanceolata, the pericarp and flesh are edible while its seed is discarded. Meanwhile, only the flesh of B. macrocarpa is edible while the other part, pericarp and seed are discarded [16]. B. lanceolata bears thick skin, white arils, with a Antioxidants 2014, 3 518 tasty, sour flavor which is common in the wild and unknown in cultivation while the B. macrocarpa have tangy sweet taste, white arils around the seeds is uncommon in the wild and already introduced in garden cultivation [17]. Plants of Baccaurea species have been shown to contain diverse phytochemicals and pharmacological properties [16,18–20]. The present study was conducted to investigate the potential of B. lanceolata and B. macrocarpa fruits as a new source of natural bioactive phytochemicals and nutraceutical. 2. Materials and Methods 2.1. Plant Material and Sample Preparation The whole fruits of B. macrocarpa and B. lanceolata were collected from Beaufort, Sabah, Malaysia. The fruits were cleaned and separated into pericarp, flesh and seed. Authentication of the fruits was done by Mr Johnny Gisil from Institute for Tropical Biology and Conservation of Universiti Malaysia Sabah, Malaysia. The pericarp, flesh and seed of the fruits were freeze-dried and ground to a fine powder. The samples were then kept in air-tight container and stored in freezer with temperature of −20 °C. 2.2. Extraction All samples were extracted using 80% methanol (at a ratio of 1:20) at room temperature on an orbital shaker for 12 h at 200 rpm [21]. The resulting slurry was filtered through a Whatman No. 1 filter paper and subsequently used for determination of total phenolic, flavonoid, anthocyanin and carotenoid contents as well as total antioxidant activity. 2.3. Determination of Total Phenolic Content (TPC) Total phenolic content was determined spectrophotometrically using Folin-Ciocalteu’s reagent as described by Velioglu et al. [21]. One hundred micro liters of sample extract was mixed with 750 μL of Folin-Ciocalteu reagent solution. The solution was mixed well using vortex and then allowed to stand at room temperature for 5 min. About 750 μL of sodium bicarbonate solution was then added to the mixture. The solution again allowed to stand at room temperature for 90 min. After 90 min, absorbance was measured at 725 nm using spectrophotometer. Gallic acid was used as standard. Total phenolic contents of the extracts were determined from the standard graph. The results were expressed as mg gallic acid equivalents in 1 g of sample (mg GAE/g). 2.4. Determination of Total Flavonoid Content (TFC) Total flavonoid content was determined using aluminum chloride spectrophotometry assay as described by Zhishen et al. [22]. About 1 mL of sample extract was added to a beaker. Four (4) mL of distilled water were then added to the mixture followed by 0.3 mL of 5% sodium nitrite. After 6 min, 0.6 mL 10% AlCl3 was added and allowed to stand for 5 min. Then, 2 mL 1M NaOH and 2.1 mL of distilled water were added to the mixture and mixed using vortex. Absorbance of the mixture was determined at 510 nm. Catechin was used as standard. The total flavonoid contents of the extracts were Antioxidants 2014, 3 519 determined from the standard graph. The total flavonoid content of the extracts was expressed as mg catechin equivalents in 1 g of sample (mg CE/g). 2.5. Determination of Total Anthocyanin Content (TAC) The total anthocyanin content was determined by the pH-differential method as described by Giusti and Wrolstad [23] where the anthocyanin pigments undergo reversible structural transformations with a change in pH manifested by strikingly different absorbance spectra, which permits accurate and rapid measurement of the total anthocyanins. The colored oxonium form predominates at pH 1.0 and the colorless hemiketal form at pH 4.5. 0.5 mL of sample extract was mixed with 3.5 mL of 0.025 M potassium chloride buffer pH 1. The mixture were then allowed to mix properly using a vortex and allowed to stand for 15 min. After 15 min, absorbance at 515 and 700 nm was measured using spectrophotometer. The same mixture was then combined with 3.5 mL of 0.025 M sodium acetate buffer pH 4.5 and allowed to stand for 15 min. After 15 min, absorbance again was measured at 515 and 700 nm using spectrophotometer. The total anthocyanin content was expressed as mg of cyanidin-3-glucoside equivalents in 100 g of dried sample (mg c-3-gE/100 g dried sample) and was calculated using the formula as follows: The total anthocyanin content (mg/100 g of dried sample) = A × MW × DF × 1000/(e × C) (1) where A is absorbance = (A515 − A700)pH1.0 − (A515 − A700)pH4.5; MW is a molecular weight + cyanidin-3-glucoside = 449.2; DF is the dilution factor of the samples, e is the molar absorbity of cyanidin-3-glucoside = 26,900; and C is the concentration of the buffer in mg/mL.